Identification of a new variant in the YMDD motif of the hepatitis B virus polymerase gene selected during lamivudine therapy

A new hepatitis B virus variant selected during lamivudine treatment was detected, in which the methionine (rtM204) in the so-called YMDD motif in the C domain of the catalytic site of the polymerase gene was replaced by a serine (rtM204S). This
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  Downloaded from byIP: Mon, 08 Feb 2016 09:05:53 VIROLOGY Identification of a new variant in the YMDD motifof the hepatitis B virus polymerase gene selectedduring lamivudine therapy HUBERT G. M. NIESTERS, ROBERT A. DE MAN  , SUZAN D. PAS, EDWIN FRIES andALBERT D. M. E. OSTERHAUS Department of Virology and    Gastroenterology, University Hospital Rotterdam, Rotterdam, The Netherlands  A new hepatitis B virus variant selected during lamivudine treatment was detected, inwhich the methionine (rtM204) in the so-called YMDD motif in the C domain of thecatalytic site of the polymerase gene was replaced by a serine (rtM204S). This changesimultaneously resulted in a tyrosine-195 into valine variant (sY195V) in the surfaceprotein HBsAg. The detection of this YSDD variant was initially observed, after anincrease of HBV DNA levels, by sequencing of amplification products from day 586. Aspecific RFLP assay was developed that could identify 10% of YSDD-containing variantsin the virus pool, which enabled detection of this new variant virus at day 506. However,by cloning several PCR products and sequencing individual recombinant clones, themutation was first identified at day 477, before a significant increase of HBV DNA wasobserved in serum. The mutation was followed by a leucine to methionine change atposition 180 (rtL180M). The consequences of this mutation for disease management anddiagnostic strategies are discussed. Introduction Potent inhibitors of hepatitis B virus (HBV) replicationsuch as lamivudine, (-)2’,3’-dideoxy-3’-thiacytidine,have recently become available. In several clinicaltrials, lamivudine therapy resulted in a rapid decline of serum HBV DNA levels in a majority of patients [1– 5]. However, as with other antiviral agents, HBVvariants resistant to the drug may emerge during prolonged treatment [2, 4, 6–10] and, after cessationof therapy, HBV DNA serum levels may return to pre-treatment values. Even enhanced HBV replication,resulting in a hepatitis flare, has been described  [11].Resistance to lamivudine occurs in up to 39% of  patients with chronic hepatitis after 1 year of therapy.Resistance is indicated by an increase of HBV DNAlevels often followed by ALT elevation. It has beenattributed solely to variations in the so-called YMDDregion of the polymerase gene, in which the methionine(rtM204) is replaced by either valine (rtV204) or isoleucine (rtI204). The replacement by valine isaccompanied by a change of leucine (rtL180) tomethionine (rtM180) [8, 10].This paper describes the identification of a new variantin the YMDD region and discusses the possibleintermediates involved in its emergence. Implicationsfor the use of different strategies to monitor theemergence of variant viruses are discussed. Theconsensus rt domain numbering system as proposed  by Stuyver   et al  . [12] has been used to describe thevariants. This numbering system is genotype indepen-dent and allows a more standardised way of describingthe antiviral-related changes. Materials and methods  Patient and clinical samples A patient of Vietnamese origin with chronic HBVinfection was treated with lamivudine 150 mg daily for a period of 756 days. The follow-up period was up to126 days after the end of treatment (day 882). Betweendays 113 and 125, interferon was added (7 days with10 MU and 5 days with 5 MU) but this was withdrawn because of side-effects. All serum samples were stored frozen at   20 8 C until use. Received 15 June 2001; revised version accepted 11 March2002.Corresponding author: Dr H. G. M. Niesters ( J. Med. Microbiol. — Vol. 51 (2002), 695–699 # 2002 Society for General MicrobiologyISSN 0022-2615  Downloaded from byIP: Mon, 08 Feb 2016 09:05:53  Measurement of HBV DNA in serum Where necessary, serum samples were diluted in knownHBV DNA-negative serum. For the accurate measure-ment of HBV DNA in serum, the Digene Hybrid Capture plate assay and an in-house TaqMan assaywere used  [13, 14]. The Digene assay enabled theaccurate determination of HBV DNA above200000 copies = ml, while the TaqMan-based assayenabled accurate determination down to 373 copies = mlwithout a sample concentration step. Differences inHBV DNA load greater than three-fold were consid-ered significant. All data were related to the EUROHEPHBV DNA standards [15]. Sequence analysis A selected genomic region of the polymerase gene wasamplified  and  sequenced with primers described  previously [16]; 2-   l amplicons were amplified withthe DYEnamic TM terminator cycle sequencing kitaccording to the manufacturer’s protocol (AmershamPharmacia, Roosendaal, The Netherlands). The PCR  products were precipitated with 3  M  NaAc, pH 5.3,ethanol 96% and centrifuged for 1 h at 2500  g   at roomtemperature. Subsequently the pellets were washed withethanol 70% and resuspended in 4   l of loading dye.The products were analysed on the ABI 373 sequencer (Applied Biosystems, Nieuwerkerk a/d IJssel). Selected PCR products were cloned into the pCRII TA vector (InVitrogen, Leek, The Netherlands). After selection of clones, a PCR reaction with the primers as above was performed directly on the recombinant colonies.  RFLP analysis of YSDD To develop a more specific screening assay for theYSDD variant, a restriction fragment length poly-morphism (RFLP) assay was developed. This detectsnucleotide changes in the HBV polymerase with therestriction endonuclease SfcI (CT . PuPyAG; NewEngland Biolabs, Beverly, MA, USA). The specificendonuclease site was introduced with a modified  primer in the PCR (sense cac.tgt.ytg.gct.agc.tat; anti-sense, generating an amplifi-cation product of 120 bp. In case of a YSDD variant, afragment of 105 bp was detected by Metaphor agarose3% gel electrophoresis with Gelstar 0.01% as inter-calating dye. Results Clinical monitoring  Routine monitoring of patients receiving lamivudinewas performed by HBV DNA quantitative assays and ALT measurements. Increased serum HBV DNA levelsare indicative of resistance to lamivudine. In Fig. 1, theHBV DNA measurements and the ALT levels aredocumented from a chronically infected patient receiv-ing lamivudine 150 mg daily for a period of 756 days.ALT values were within the normal range during and after therapy, except when interferon was added. Themaximal antiviral effect of lamivudine was a 5.11 log 10 HBV DNA decrease down to 10000 copies = ml at day244. HBV DNA levels remained at 4–4.77 log 10  between days 127 and 378. A 0.5 log 10  increase of HBV DNA was observed at day 506 compared withday 441, after which levels increased to 8.31 log 10  atday 756. After the end of therapy, HBV DNA levelsrose to 8.85 log 10 , similar to the pre-treatment value. Sequence analysis Sequencing of PCR products at days 586 and 623identified a change of rtM204S, together with anrtL180M change. At day 506, only YMDD wild-typesequences were found but day 532 both rtM204 and rtS204 were detected simultaneously. The methionineto serine change was the result of a two nucleotidechange of ATG into AGT, thereby also introducing atryptophan to valine substitution at position 195 of theHBsAg (sY195V, TGG changed to GTG). The viruswas typed  as HBV genotype B ( ayw 1 serotypeaccording to [17]). Interferon    R   F   L   P   S  e  q  u  e  n  c   i  n  g   S   T   O   P 345678910    H   B   V   D   N   A   l  o  g         1        0    g  e  q   /  m   l 0 100 200 300 400 500 600 700 800 900 1000Days after start therapy050100150200250300    A   L   T   I   U   /  m   l Fig. 1.  HBV DNA and ALT levels in a patient with a confirmed rtS204 variant in the polymerase gene (C domain). The patientreceived lamivudine 150 mg/day for 756 days. Days on which the rtS204 sequence was confirmed by analysis of PCR product or RFLP analysis are indicated by arrows. 696 H. G. M. NIESTERS  ET AL .  Downloaded from byIP: Mon, 08 Feb 2016 09:05:53 As sequencing of PCR products by itself is not able todetect minor variants, amplification products generated  between days 477 and 532 were cloned and up to 40independent clones per time point were sequenced. Atday 477, 95% of the clones sequenced contained wild-type rtM204 sequences, 2.5% of the clones contained only the rtS204 change, and 2.5% of the clonescontained both rtS204 and rtM180. At day 506, 72.4%of the clones contained wild-type rtM204 sequences,24.1% contained the rtM180 and rtS204 changes and 3.4% contained only the rtS204 change. At day 532,these percentages were 72% for wild-type rtM204sequences, 20% for both rtM180 and rtS204 changes,and 8% for only the rtS204 change. After day 532,100% variant sequences rtM180 and rtS204 weredetected.  RFLP analysis To enable easy and early detection of YSDD variants, aspecific RFLP assay was developed with the restrictionendonuclease  Sfc I and a modified amplification primer.The RFLP assay had a detection limit down to 1000copies/ml, with the EUROHEP HBV DNA standard asreference, and enabled the detection of 10% YSDD-containing variant virus in a total HBV population(data not shown).RFLP analysis of samples from selected dates duringand after treatment was performed (Fig. 2). The firstindication for the presence of a YSDD variant was aminor band representative of this variant obtained atday 506. At day 596, the minor HBV populationcontained wild-type sequences. At day 623 only YSDDvariant sequences could be detected until the end of treatment (day 756). Wild-type sequences were alreadydetected 13 days after the end of treatment. Surpris-ingly, until the last sample analysed at day 882, bothYSDD and YMDD sequences were detected in equalamounts. No return to wild-type sequences only wasseen. Discussion A new HBV variant isolated from a patient with achronic hepatitis B virus infection has been described.In this variant, which was genotypically characterised during prolonged treatment with lamivudine, thertM204 of the YMDD domain in the polymerase genewas changed into rtS204. The data on the rtS204variant confirm the presence of this variant duringlamivudine treatment in the patient, but do not prove anactual reduced drug susceptibility. The two previouslydescribed mutations in this domain are known asrtM204I and rtM204V, both of which result in reduced replication of HBV [18].We and others have shown that mutations related tolamivudine arise during prolonged treatment, both intransplant patients and in other chronically infected HBV patients [4, 7, 10, 19, 20]. However, the incidenceand patterns with which they occur may be different inthe populations studied. It has been demonstrated clearly that the major mutation responsible for resistance is located in the C domain of the catalyticYMDD site of the polymerase gene. Furthermore, asecond mutation rtL180M in the B domain of the polymerase is often linked to the particular rtM204Vchange. However, this correlation has also beendescribed recently in a limited number of patients withthe rtI204 variant [6, 7].There are accumulating data from molecular modellingand biochemical characterisation of variants that givemore detailed insight into the mechanism of resistance of HBV and HIV-1 to lamivudine [21– 23]. Steric hindrance between the side chains of the   -branched amino acids valine, isoleucine and threonine in theYMDD motif with the oxathiolane ring of lamivudinemost likely accounts for the phenotypic resistance of   both viruses [22, 23]. This model may be a simplifica- tion, as in-vitro studies have shown that, for example,YADD variants of HIV-1 and Moloney murineleukemia virus are also resistant to lamivudine [21].These authors suggest that other structural changes of the polymerase active site due to the mutation or therepositioning of the complex between the polymerase,the nucleic acid and the dNTP binding site eventuallylead to steric hindrance. The described rtS204 variantof HBV would most likely cause changes in thisreplication complex according to this model. Fig. 2.  RFLP analysis for the rapid detection of the rtS204 variant in the polymerase gene of HBV. PCR primers were designed toamplify a region of 120 bp. After digestion with the restriction endonuclease  Sfc I, a 15-bp fragment is removed from YSDDvariants, leaving a 105-bp fragment. M, marker line (no signal).  NEW VARIANT IN YMDD MOTIF CO HEPATITIS B VIRUS 697  Downloaded from byIP: Mon, 08 Feb 2016 09:05:53 The YADD and YSDD mutants of HIV-1 are replica-tion competent  in vitro , but have not been observed   invivo  in HIV-1-infected individuals [24]. Proviruses withreverse transcriptase containing these mutations wereless infectious than wild-type virus. All reported variants related to lamivudine resistance selected   invivo  so far have been caused by a single nucleotidechange, whereas two mutations are required to convertthe methionine codon into an alanine or serine codon.The change of methionine into serine in the HBV-infected patient in the present study is due to thechange of ATG into AGT. As a working hypothesis itwas assumed that a YIDD intermediate with the codingsequence ATT should precede the occurrence of theYSDD variant. However, despite screening of a largenumber of individual recombinant clones, this inter-mediate was not found.The present study also addressed the time course of changeover from wild-type into variant sequence, aswell as the events that occurred after the end of therapy. It was expected that after the end of therapy,wild-type sequences would be detected within a monthwithout the detection of variant sequences. However,wild-type as well as variant viruses were detected inalmost equal frequencies from day 13 onward until thelast sample analysed 3 months later. A plausibleexplanation for this observation is that the replicativeintermediate, known as covalently closed circular DNA(cccDNA), of HBV containing the variant sequenceshas established itself in the liver. As this cccDNA isnot inhibited by lamivudine, the variant virus may haveinfected a large proportion of hepatocytes, as indicated  by the high viral load of variant virus present at theend of treatment.From a diagnostic point of view, the question arises asto how these variants can be predicted or identified aseasily and quickly as possible. Assuming that thevariant strains replicate less efficiently than wild-typevirus, but better than the maximally suppressed virus population, accurate and well standardised viral load measurements should be indicative. Use of both acommercially available assay as well as an in-houseassay demonstrated this phenomenon. Furthermore,easy-to-use methods to identify viral strains have been proposed, such as the RFLP method and the InnoLipaHBV reverse hybridisation assay [25, 26]. Both meth-ods seem to be sensitive, but can be used only for theidentification of known variants, which is the crucial problem. We have been sequencing routinely variant populations even though this lacks sensitivity. However,it is the only method currently available which enablesidentification of new mutations that could be related toresistance, in combination with accurate viral DNAmeasurements.In conclusion, the present study has shown for the firsttime the detection of a new variant of HBV selected under lamivudine treatment. This YSDD variant wasalso linked with the rtL180M substitution. The popu-lation of viruses present in circulation after the end of therapy contained both wild-type and variantsequences. Furthermore, these findings suggest thatthe use of different techniques to screen for thesevariants should always include sequence analysis. It is possible that more variants will be found to ariseduring treatment with lamivudine. This work was partly supported by a Revolving Fund Grant from theUniversity Hospital Rotterdam. Accession numbers for sequences areAY033072 and AY033073. References 1. 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